LSH10 Antibody

Basic Research Questions

How to validate LSH10 antibody specificity for chromatin immunoprecipitation (ChIP)?

• Methodological guidance:

  • Perform loss-of-function mutant controls (e.g., lsh10-1 and lsh10-2 lines) to confirm antibody signal depletion in mutants compared to wild-type plants .

  • Use transgenic complementation lines (e.g., lsh10-1/LSH10-His6) to verify antibody recognition of epitope-tagged LSH10 .

  • Validate via EMSA competition assays: Compare binding patterns of recombinant GST-LSH10 with plant-extracted LSH10-antibody complexes to confirm target sequence recognition .

What experimental systems are optimal for studying LSH10-OTLD1 interactions?

• Key approaches:

  • Bimolecular Fluorescence Complementation (BiFC): Confirms nuclear localization of LSH10-OTLD1 complexes .

  • Fluorescence Resonance Energy Transfer (FRET): Validates proximity (<10 nm) between LSH10 and OTLD1 in live cells .

  • Co-immunoprecipitation (Co-IP): Use Myc/FLAG-tagged constructs in Arabidopsis protoplasts with anti-LSH10 antibodies for pulldown assays .

How to distinguish LSH10 from other ALOG family members in antibody-based assays?

• Resolution strategies:

  Parameter	LSH10 Specificity	Cross-Reactivity Check
  Epitope	C-terminal His6 tag	Test against LSH1/LSH3/LSH4 mutants
  DNA binding	YACTGTW motif	Compare EMSA shifts with LSH1/LSH3 probes
  Mutant phenotype	20–60x upregulation of OSR2, WUS, ABI5, ARL	Monitor GA20OX as negative control

Advanced Research Questions

How to resolve conflicting data on H2B deubiquitylation levels in lsh10 mutants?

• Contradiction analysis framework:

  • Chromatin context: Compare qChIP results across target genes (OSR2: 4.08x vs. ARL: 60.2x H2Bub1 increase) .

  • Time-course experiments: Assess temporal changes in H2Bub1 during seedling development.

  • OTLD1 redundancy: Test double mutants (e.g., lsh10 otld1) to identify compensatory mechanisms .

What optimization is required for EMSA with LSH10 antibodies?

• Technical adjustments:

  Variable	Optimal Condition	Rationale
  Probe length	25–35 bp (Fig. 5A )	Maintains motif integrity while avoiding nonspecific binding
  Competitor DNA	50–100x molar excess unlabeled probe	Resolves fuzzy DNA recognition patterns
  Antibody conc.	1:200 for supershift assays	Balances signal clarity and complex stability

How to design multiplex experiments studying LSH10’s role in transcriptional repression?

• Integrated workflow:

  • Phase 1: RNA-seq on lsh10 mutants to identify deregulated genes (e.g., 21.4x OSR2 upregulation ).

  • Phase 2: AmpDAP-seq to map LSH10-binding peaks (Dataset S1B ).

  • Phase 3: H2Bub1 qChIP to link chromatin state with transcriptional output .

Data Reliability and Reproducibility

How to address variability in LSH10 antibody performance across plant tissues?

• Troubleshooting matrix:

  Issue	Solution	Reference
  Low ChIP signal	Use young inflorescence tissues (<3 mm buds)	Fig. 2A
  Cross-reactivity	Pre-absorb antibodies with lsh1/lsh3 mutant extracts	SI Appendix, Fig. S14
  Background noise	Include ga20ox as non-target promoter control	Fig. 4B

What orthogonal methods validate LSH10 antibody specificity in Arabidopsis?

• Multilevel validation:

  • Genetic: Compare lsh10 mutants vs. complemented lines (Fig. 4C ).

  • Biochemical: Immunoblotting with truncated LSH10ΔC variants .

  • Genomic: Cross-reference ampDAP-seq peaks with RNA-seq deregulated genes (Dataset S1D ).